過敏是全世界最常見的疾病之一，罹患過敏的人口也是逐年增加，因此過敏的快速檢測具有相當的重要性。目前臨床上大都檢測免疫球蛋白IgE定性的來判知患者屬何種原因所引發的，但迄今被歸類的四種過敏反應，本質上乃因嗜鹼性白血球細胞或肥大細胞受到活化或刺激後，分泌了組織胺、白三烯素等化學物質，導致急、慢性的紅腫熱痛，故若能針對造成過敏反應的物質作檢測是最直接且必要的方法。本研究的電化學式毛細管電泳(CE-EC )晶片，乃藉由毛細管電泳可快速分離不同物質以及濃縮樣本的優勢，更搭配具即時性且高靈敏度的電化學偵測法，期望達到對組織胺樣本作即時性的檢測。晶片是以聚甲基矽氧烷(PDMS)構成注入與分離之微管道(注入管道是採用雙T型設計，寬為100 m)，此PDMS微管道與已製作有電極組的載玻片，兩者均施予氧電漿處理後，面對面對準接合而成。所製作的檢測電極組以End-channel模式來配置，亦即將金工作電極(寬度100 m)置於管道的出口端，參考電極則是以循環伏安法電鍍之白金參考電極(寬度100 m)，輔助電極與接地電極則是使用白金線。晶片微管道的電滲流遷移率量測結果約為3.5~4.0×10-4 cm2/Vs，且在晶片上以循環伏安法掃描組織胺樣本在+0.7~+0.9 V (vs. Pt reference)可觀測到其氧化電流，故實驗中的電位皆固定在+0.9 V。組織胺在10 mM PB (pH 7.0)溶液中最佳的電泳實驗檢測條件為注入電場+200 V/cm、注入時間15 s、分離電場+160 V/cm以及+600 V之推回電壓，此條件下組織胺線性範圍為10 M-100 M (R2=0.989)，檢測極限為2.5 M (S/N=3)，訊號到達時間為46.9±1.7 s。最後，本系統也可成功分離組織胺與組織胺酸兩種物質，並可初步測得KU-812細胞(7×108 cells/mL)經10 M鈣離子載體(A23187)刺激20 min後，平均一顆細胞組織胺分泌量約67 amol，訊號出現時間約在65 s，未來本系統可望能再被以細胞級的程度應用於臨床上細胞過敏反應的評估。

Allergic reaction is one of the universal diseases around the world, and the number of patients suffering from allergic reaction is increasing year by year. Thus, immediate diagnosis of allergies is very important. Unlike the clinical diagnosis of allergies by qualitative determination of IgE level, detection of the allergic analyte like histamine or leucotriene and etc, released from basophils or mast cells should be a direct method. The CE-EC chips in our study have the advantages of high-speed separation, sample stacking, and high sensitivity, resulting in a convenient tool for histamine detecting. We constructed the injection and separation channels with polydimethylsiloxane (PDMS). A 100 m double-T design was employed for the injection channel. The chips were modified and bound by O2 plasma treatment. The arrangement of electrodes in our chips was end-channel mode, while the Au working electrode (100 m) was positioned just outside the separation channel. The Pt reference electrode (100 m) was fabricated by cyclic voltammetry scanning for Pt-deposition and the counter and grounding electrodes were Pt wires. In EOF measurement, we found that the EOF mobility (EOF) in PDMS channel was around 3.5~4.0×10-4 cm2/Vs. We performed cyclic voltammetry on our chip and found that histamine was oxidized on the Au electrode at +0.7~+0.9 V (vs. Pt reference). Therefore, we selected +0.9 V for our detection voltage. In capillary electrophoretic measurements, the optimum conditions for histamine detection in 10 mM PB (pH 7.0) were +200 V/cm injection field, 15 s injection time, +160 V/cm separation field, and +600 V pushback voltage. Under these conditions, histamine responded linearly from 10 M to 100 M (R2=0.989) and the limit of detection was 2.5 M (S/N=3). The retention times of histamine signals were at 46.9±1.7 s. Furthermore, histamine and histidine could be separated successfully in our microchip. In preminary result, we also detected the histamine released from the KU-812 cells (7×108 cells/mL) which were stimulated 20 min by 10 M calcium ionophore (A23187). The mean amount of histamine was in ca. 67 amol/cell. The retention times of histamine signals were at 65 s. In the future, this resulting system could be applied for clinical investigations and allergy diagnosis in cells level.

[1] P. Gell, Coombs RRA, eds. Clinical Aspects of Immunology. 1st ed. Oxford, England: Blackwell, 1963
[2] S. Tanaka, “Physiological function mediated by histamine synthesis”, Yakugaku Zasshi, 123 (2003) 547-559
[3] L. Maintz, T. Bieber, N. Novak, “Histamine Intolerance in Clinical Practice”, Dtsch Artzebl, 103 (2006) A3477-A3483
[4] http://fig.cox.miami.edu/~lfarmer/BIL265/immunesystem.htm
[5] http://microvet.arizona.edu/courses/mic419/Secure/CaseAtopicDerm/TypeIhypersens.htm
[6] A. Falus, “Histamine: biology and medical aspects”, Hungary: SpringMed Publishing, (2004) 43–52
[7] L. Maintz, N. Novak, “Histamine and histamine intolerance”, American Journal of Clinical Nutrition, 85 (2007) 1185-1196
[8] T. Toyo’oka, “Separation assay of histamine and its metabolites in biological specimens”, Biomedical Chromatography, 22 (2008) 919-930
[9] K. Pittertschatscher, R. Hochreiter, J. Thalhamer, P. Hammerl, “Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography–mass spectrometry method”, Analytical Biochemistry, 308 (2002) 300-306
[10] J. Y. Hui, S. L. Taylor, “Reversed-phase ion-pair high-performance liquid-chromatographic procedure for determination of histamine and its metaboltes in rat urine”, Journal of Chromatography, 312 (1984) 443-449
[11] N. Kitanaka, J. Kitanaka, M. Nishiguchi, H. Kinoshita, H. Ouchi, T. Minami, S. Hishida , M. Takemura, “Decreased histamine stimulated phosphoinositide hydrolysis in the cerebral cortex of a rat line selectively bred for high alcohol preference”, Neurochemical Research, 29 (2004) 1431-1436.
[12] H. Xinqiang, M. M. Vohra,“A sensitive method for simultaneous determination of histamine and noradrenaline with high-performance liquid chromatography/electrochemistry”, Journal of Pharmacological Methods, 25 (1991) 29-40
[13] T. Toyo’oka, “Modern derivatization methods for separation sciences”, Wiley: Chichester, 1999
[14] A. Yamatodani, H. Fukuda, H. Wada, T. Iwaeda, T. Watanabe, “High-performance liquid-chromatographic determination of plasma and brain histamine without previous purification of biological samples-cation-exchange chromatography coupled with post-column derivatization fluorometry”, Journal of Chromatography, 344 (1985) 115-123
[15] K. Saito, M. Horie, N. Nose., “High performance liquid-chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization”, Journal of Chromatography, 595 (1992) 163-168
[16] http://coa.cpc.org.tw (張菊香, 水產品中組織胺的生成及檢測方法之探討)
[17] T. Yoshitake, F. Ichinose, H. Yoshida, K. Todoroki, J. Kehr, O. Inoue, H. Nohta, M. Yamaguchi, “A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection”, Biomedical Chromatography, 17 (2003) 509-516
[18] F. Nishiwaki , K. Kuroda, Y. Inoue, G. Endo, “Determination of histamine, 1-methylhistamine and N-methylhistamine by capillary electrophoresis with micelles”, Biomedical Chromatography, 14 (2000) 184-187
[19] O. Shigeyuki, Y. Yukari, M. Makiko, F. Yuhei, O. Koji, T. Shigeru, “Selective detection of biogenic amines using capillary electrochromatography with an on-column derivatization technique”, Analytical Chemistry, 74 (2002) 3463-3469
[20] http://www.oxfordbiomed.com “Enzyme immunoassay for histamine (Life Science Format) (Product No. EA 31)”, Oxford Biomedical Research
[21] K. Yamamoto, K. Takagi, K. Kano, T. Ikeda, “Bioelectrocatalytic detection of histamine using quinohemoprotein amine dehydrogenase and the native electron acceptor cytochrome c-550”, Electroanalysis, 13 (2001) 375-379
[22] Y. Matsubara, Y. Murakami1, M. Kobayashi, Y. Morita, E. Tamiya, “Application of on-chip cell cultures for the detection of allergic response”, Biosensors and Bioelectronics, 19 (2004) 741-747
[23] Q. Weng, F. Xia, W. Jin, “Measurement of histamine in individual rat peritoneal mast cells by capillary zone electrophoresis with electrochemical detection”, Journal of Chromatography B, 779 (2002) 347-352
[24] F. Foret, L, Krivankova, P. Bocek, “Capillary zone electrophoresis”, John Wiley & Sons, (1993) 37-94
[25] D. S. Burgi, R. L. Chien, “Optimization of sample stacking for high performance capillaryelectrophoresis”, Analytical Chemistry, 63 (1991) 2042-2047
[26] R. Bharadwaj, J. G. Santiago, “Dynamics of field-amplified sample stacking”, Journal of Fluid Mechanics, 543 (2005) 57-92
[27] G. Hempel, “Strategies to improve the sensitivity in capillary electrophoresis for analysis of drugs in biological fluids”, Electrophoresis, 21 (2000) 691-698
[28] H. F. Li, Z. Cai, J. M. Lin, “Separation of catecholamines by microchip electrophoresis with a simple integrated laser-induced fluorescence detector”, Analytica Chimica Acta, 565 (2006) 183-189
[29] 蔡有光 “質譜儀技術於蛋白質體分析之應用”, 永進生物科技股份有限公司
[30] W. R. Vandaveer IV, S. A. Pasas-Farmer, D. J. Fischer, C. N. Frankenfeld, S. M. Lunte, “Recent developments in electrochemical detection for microchip capillary electrophoresis”, Electrophoresis, 25 (2004) 3528-3549
[31] X. Huang, T. J. Pang, M. J. Gordon, R. N. Zare, “On-column conductivity detector for capillary zone electrophoresis”, Analytical Chemistry, 59 (1987) 2747-2749
[32] J. Ye, R. P. Baldwin, “Amperometric detection in capillary electrophoresis with normal size electrodes”, Analytical Chemistry, 65 (1993) 3525-3527
[33] R. W. Cattrall, H. Freiser, “Coated wire ion-selective electrodes”, Analytical Chemistry, 43 (1971) 1905-1906
[34] D. J. Fischer, M. K. Hulvey, A. R. Regel, S. M. Lunte, “Amperometric detection in microchip electrophoresis devices: effect of electrode material and alignment on analytical performance”, Electrophoresis, 30 (2009) 3324-3333
[35] X. Huang, R. N. Zare, S. Sloss, A. G. Ewing, “End-column detection for capillary zone electrophoresis”, Analytical Chemistry, 63 (1991) 189-192
[36] R. S. Martin, K. L. Ratzlaff, B. H. Huynh, S. M. Lunte, “In-channel electrochemical detection for microchip capillary electrophoresis using an electrically isolated potentiostat”, Analytical Chemistry, 74 (2002) 1136-1143
[37] Y. Kong, H. Chen, Y. Wang, S. A. Soper, “Fabrication of a gold microelectrode for amperometric detection on a polycarbonate electrophoresis chip by photodirected electroless plating”, Electrophoresis, 27 (2006) 2940-2950
[38] Y. Wang, H. Chen, Q. He, S. A. Soper, “A high-performance polycarbonate electrophoresis microchip with integrated three-electrode system for end-channel amperometric detection”, Electrophoresis, 29 (2008) 1881-1888
[39] N. A. Lacher, S. M. Lunte, R. S. Martin, “Development of a microfabricated palladium decoupler/electrochemical detector for microchip capillary electrophoresis using a hybrid glass/poly (dimethylsiloxane) device”, Analytical Chemistry, 76 (2004) 2482-2491
[40] C. C. Wu, R. G. Wu, J. G. Huang, Y. C. Lin, H. C. Chang, “Three-electrode electrochemical detector and platinum film decoupler integrated with a capillary electrophoresis microchip for amperometric detection”, Analytical Chemistry, 75 (2003) 947-952
[41] H. Stark, “Histamine receptors”, Biotrend Reviews, No.01, November 2007
[42] A. Plecis, Y. Chen, “Improved glass–PDMS–glass device technology for accurate measurements of electro-osmotic mobilities”, Microelectronic Engineering, 85 (2008) 1334-1336
[43] M. Castano-Alvareza, M. T. Fernandez-Abedula, A. Costa-Garcia, M. Agirregabiria, L. J. Fernandezb, J. M. Ruano-Lopezb, B. Barredo-Presac, “Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters”, Talanta, 80 (2009) 24-30
[44] 莊景棋, 以ITO電極發展電化學式毛細管電泳晶片於亞硝酸根之檢測, 國立成功大學醫學工程研究所碩士論文, 2008
[45] H. Makamba, J. H. Kim, K. Lim, N. Park, J. H. Hahn, “Surface modification of poly(dimethyl siloxane) microchannels”, Electrophoresis, 24 (2003) 3607-3619
[46] X. Huang, M. J. Gordon, R. N. Zare, “Current-monitoring method for measuring the electroosmotic flow rate in capillary zone electrophoresis”, Analytical Chemistry, 60 (1988) 1837-1838
[47] J. C. Foreman, J. L. Mongar, B. D. Gomperts, “Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process”, Nature, 245 (1973) 249-251
[48] J. J. Whalen III, J. D. Weiland, P. C. Searson, “Electrochemical deposition of platinum from aqueous ammonium hexachloroplatinate solution”, Journal of the Electrochemical Society, 152 (2005) C738-C743
[49] H. Wubke, R. Jan, T. D. Thanh, R. Robert, A. Dario, R. Alexandra, “Poly (oxyethylene) based surface coatings for poly(dimethylsiloxane) microchannels’’, Langmuir, 21 (2005) 7551-7557
[50] M. Morra, E. Occhiello, R. Marola, F. Garbassi, P. Humphrey, D. Johnson,“On the aging of oxygen plasma-treated polydimethylsiloxane surfaces”, Journal of Colloid and Interface Science, 137 (1990) 11-24
[51] C. H. Lin, R. J. Yang, C. H. Tai, C. Y. Lee, L. M. Fu, “Double-L injection technique for high performance capillary electrophoresis detection in microfluidic chips”, Journal of Micromechanics and Microengineering, 14 (2004) 639-646
[52] P. Ertl, C. A. Emrich, P. Singhal, R. A. Mathies, “Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system”, Analytical Chemistry, 76 (2004) 3749-3755
[53] F. Matysik, “Improved end-column amperometric detection for capillary electrophoresis”, Journal of Chromatography A, 742 (1996) 229-234
[54] K. Pihel, S. Hsieh, J. W. Jorgenson, R. Mark Wightman, “Quantal corelease of histamine and 5-hydroxytryptamine from mast cells and the effects of prior incubation”, Biochemistry, 37 (1998) 1046-1052
[55] D. P. Manica, Y. Mitsumori, A. G. Ewing, “Characterization of electrode fouling and surface regeneration for platinum electrode on an electrophoresis microchip”, Analytical Chemistry, 75 (2003) 4572-4577
[56] 賴冠宇, 以電化學式微流體晶片之胞吐組織胺的線上監測, 國立成功大學醫學工程研究所碩士論文, 2010
[57] 吳佳儀,可供組織胺檢測的毛細管電泳晶片電化學晶片之研發, 國立成功大學醫學工程研究所碩士論文, 2005